Newly identified human small intestine extracellular lactase-phlorizin hydrolase (ecLPH) gene

ABSTRACT

The invention provides a transgenic animal producing low-lactose milk, which is transformed with a gene encoding an extracellular lactase-hydrolyzing enzyme cloned from a human small intestinal cDNA library. The invention also provides a new extracellular lactase-phlorizin hydrolase (ecLPH) gene that can express human lactase-hydrolyzing enzyme in the mammary gland of animals. The invention can be used in the production of low-lactose milk.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/434,332, filed May 15, 2006, the contents of which arehereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the biotechnical field of gene cloningand transgenic animals, which utilizes a genetic cloning techniqueinvolving the human genomic library, and development of mammarygland-specific expression vectors to provide a platform technology oftransgenic animals producing low-lactose milk. More specifically, thepresent invention also provides a new extracellular lactase-phlorizinhydrolase (ecLPH) gene, which can be used to transform animals toproduce low-lactose milk.

BACKGROUND OF THE INVENTION

Milk is a food source of high nutrition value, which is rich inproteins, vitamins, carbohydrates, calcium and other minerals. Exceptfor being utilized as the nutrition source for newborn babies andinfants, it can also be used to prevent osteoporosis in older people,and especially elderly people. Lactose is the major carbohydrate inmilk. However, people with lactose intolerance cannot decompose theabundant lactose in milk, which is caused by reduction in the activityof lactase-phlorizin hydrolase. Un-decomposed lactose accumulates in theintestine and induces bacterial fermentation, producing carbon dioxide,hydrogen and methane and resulting in symptoms such as abdominaldistension, nausea and diarrhea. Such disadvantages limit the use ofmilk and dairy products including milk or lactose.

The best strategy to treat patients suffering from lactose intolerancesyndrome or to prevent the syndrome, is to avoid the ingestion of dairyproducts containing large amounts of lactose. However, many people,including lactose-intolerant people, enjoy dairy products containingmilk or otherwise containing lactose. Therefore, the development oflow-lactose milk and other dairy products containing lactose is crucial.It is now known that α-lactalbumin is an abundant calcium metalloproteinin milk. The binding between α-lactalbumin and galactosyltransferase inthe Golgi body can modify the specificity of the galactosyltransferaseby forming the lactose synthetase binary complex to synthesize lactose.In order to understand the effect of α-lactalbumin on lactogenesis andthe relationship between α-lactalbumin and lactose, Stinnakre et al.utilized gene targeting in embryonic stem cells to produce transgenicmice with heterozygous or homozygous deficiencies in the α-lactalbumingene. They found that the milk of the transgenic mice with homozygousdeficiencies in the α-lactalbumin gene lacked not only α-lactalbumin butalso lactose. However, the lack of lactose resulted in highly viscousmilk since lactose is an important regulator of osmotic pressure duringlactation, and thus the female mice cannot feed their pups smoothly. Inthe milk of the transgenic mice with heterozygous deficiencies in theα-lactalbumin gene, α-lactalbumin decreased by 40% while lactosedecreased by 10-20% (Stinnakre, M. G., Vilotte, J. L., Soulier, S. andMercier, J. C., 1994. Creation and phenotype analysis ofα-lactalbumin-deficient mice. Proc. Natl. Acad. Sci. 91: 6544-6548).

In 1996, L'Huillier et al. created a construct wherein a ribozyme 5(RZ5) gene is downstream of the mouse mammary tumor virus (MMTV) longterminal repeat (LTR) so that the RZ5 gene can be specifically expressedin mammary gland cells. Utilizing gene-transformation technology,transgenic mice highly expressing the RZ5 gene in their mammary glandswere developed. RZ5 can specifically degrade α-lactalbumin mRNA toreduce the production of α-lactalbumin and further affects theproduction of lactose (L'Huillier, P. J., Soulier, S., Stinnakre, M. G.,Lepourry, L., Davis, S. R., Mercier, J. C. and Vilotte, J. L., 1996.Efficient and specific ribozyme-mediated reduction of bovinealpha-lactalbumin expression in double transgenic mice. Proc. Natl.Acad. Sci. 93: 6698-6703). According to the studies of Berns andHauswirth (1979), adeno-associated virus (AAV), which istissue-specific, uses cells in the tissues of human respiratory andgastrointestinal systems as host cells. Moreover, since AAV is resistantto high temperature and low pH, it is potentially resistant to oraladministration (Bern, K. I. and Hauswirth, W. W., 1979. Adeno-associatedviruses. Adv. Virus. Res. 25: 407-409). In 1998, During et al.reconstructed AAV into a vector, wherein most of the AAV genes weredeleted and only 145 bp of terminal repeats were retained. The resultingAAV vector contains few viral genes so that the possibilities of generecombination and viral gene expression were reduced to minimum. Then aβ-galactosidase gene was constructed onto the AAV vector, and the vectorwas fed to rats through oral administration. It is found that theβ-galactosidase gene was highly expressed at the 6th hour post feeding,and the expression was sustained and stable through 6 months (During, M.J., Xu, R., Young, D., Kaplitt, M. G., Sherwin, R. S. and Leone, P.,1998. Peroral gene therapy of lactose intolerance using anadeno-associated virus vector. Nature Medicine 4: 1131-1135).

Even in view of the aforementioned studies, acceptable low-lactose milkhas not been developed. In one aspect, the normal osmotic pressure oflactation is affected. In another aspect, the use of viral vectors haspotential danger. Therefore, the development of a new technique forproducing low-lactose milk and other dairy products containing lactoseis necessary. The present invention satisfies this necessity.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention relates to a transgenic animalproducing low-lactose milk, which is transformed with a gene encoding anextracellular lactase-hydrolyzing enzyme, wherein the gene is clonedfrom a human small intestinal cDNA library. Preferably, the gene is agene encoding an extracellular lactase-phlorizin hydrolase (ecLPH).

Another aspect of the present invention relates to an isolated newextracellular lactase-phlorizin hydrolase gene cloned from a human smallintestinal cDNA library that can be expressed with a mammarygland-specific expression vector.

A further aspect of the present invention relates to a heterologous geneexpression vector construct, comprising the extracellularlactase-phlorizin hydrolase (ecLPH) gene of the present inventionconstructed in a mammary gland-specific expression vector which can beexpressed in a mammary gland.

Yet another aspect of the present invention relates to an isolatedextracellular lactase-phlorizin hydrolase gene cloned from a human smallintestinal cDNA library that can be expressed with a mammarygland-specific expression vector, encoding an extracellular proteinwithout a membrane-binding domain, the gene lacking Exon 12 and Exon15-17 compared with a known human lactase-phlorizin hydrolase (hLPH)gene.

A still further aspect of the present invention relates to a method forproducing low-lactose milk, wherein the heterologous gene expressionvector construct of the present invention is introduced into apronucleus to produce a transgenic animal, which produces low-lactosemilk to benefit the large group of people suffering from lactoseintolerance syndrome.

Another aspect of the present invention relates to a heterologous geneexpression vector construct comprising the gene of the present inventionthat can be expressed with a mammary gland-specific expression vector,and (1) a promoter 5′-regulatory sequence, which is specific forexpression in mammary gland epithelial cells and can regulate theheterologous gene so that the gene is continuously and stably expressedduring a lactation period of a transgenic mammal; (2) a mammarygland-specific extracellular signal peptide sequence, which can guidethe heterologous proteins expressed in the mammary gland epithelialcells of the transgenic mammal so that the proteins are efficientlysecreted into milk produced by the transgenic mammal; (3) a heterologousgene, which is downstream of and regulated by the 5′-regulatory sequenceand the extracellular signal peptide sequence; and (4) a 3′-regulatorysequence, which is downstream of the coding sequence of the heterologousgene, comprising a polyadenylation signal sequence for stability andintegrity of an mRNA molecule transcripted from the heterologous geneand post-transcriptional modification.

A further aspect of the present invention relates to a method forproducing low-lactose milk, comprising providing the transgenic animalof the present invention, and obtaining the low-lactose milk from thetransgenic animal.

Another aspect of the present invention relates to a method forincreasing the growth rate and weight gain of a newborn animal,comprising providing a maternal milk-producing animal and transformingthe animal by introducing into the animal the extracellularlactase-phlorizin hydrolase gene of the present invention, and feedingthe newborn animal with milk secreted by the maternal animal afterpregnancy and delivery of the newborn animal.

Still another aspect of the present invention relates to a method forproducing a transgenic animal that produces low-lactose milk, comprisingproviding a milk-producing animal and transforming the animal byintroducing into the animal a gene encoding an extracellularlactase-hydrolyzing enzyme cloned from a human small intestinal cDNAlibrary.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly indicates otherwise.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows the results obtained by screening the human small intestine5′-stretch cDNA library for the newly cloned extracellularlactase-phlorizin hydrolase (ecLPH) gene of the present invention. Theleft part of the drawing shows colonies of the cDNA phage clonesobtained after three rounds of screening. The right part of the drawingis the restriction digestion map of the ecLPH phage clones.

FIG. 2 shows the cDNA structure of the newly cloned extracellularlactase-phlorizin hydrolase (ecLPH) gene of the present invention(bottom panel) compared to the known human lactase-phlorizin hydrolase(hLPH) gene (top panel). The hLPH-encoded protein containing a secretionsignal peptide in the N-terminus (initial red box region in the toppanel) and a transmembrane domain (green box region in the top panel)has been reported as a membrane-anchored protein in the intestinalepithelia. However, the newly identified human ecLPH cDNA structurecontains an alternative splicing in the transmembrane domain of hLPH anda novel 3′-UTR sequence (yellow box region in the bottom panel). Thisclone was examined as a feature of the membrane-anchorless extracellularprotein in the present invention.

FIGS. 3A-3C show the results obtained by examining the ecLPH genes inthe genomes of the transgenic mice and dairy goats of the presentinvention. FIG. 3A is a map of the transgene construct. FIG. 3B showsthe results of Southern blot analysis of the transgenic mouse genomes ofdifferent lines in transgenic founder (F₀) and their offsprings (F₁)(transgenic line #3 is shown in left blot and transgenic line #5 isshown in right blot), which prove that the heterologous ecLPH gene hasindeed been inserted into the chromosomes of transgenic animals. FIG. 3Cshows the result of express PCR analysis of the transgenic mice (M1-M7)and transgenic goats (G1-G3).

FIG. 4 shows the result of Western blot analysis of the ecLPH proteinssecreted in the milk of the transgenic animals of the present invention.Milk of transgenic mice and normal mice was diluted 20-fold andcentrifuged to remove milk lipid. SDS-PAGE and Western blot analysiswere performed.

FIGS. 5A-5C show immuno-histochemistry staining of mammary gland tissuesections of the ecLPH transgenic mice of the present invention.Immuno-histochemistry staining was performed to the mammary glandtissues of transgenic mice (A and B) and normal mice (C) with monoclonalantibodies specific for LPH protein. Mammary-gland tissues were taken onthe 14^(th) day of the lactation period. Antibody staining analysisshowed that homozygous transgenic female mice (B) expressed more ecLPHproteins than heterozygous ones (A).

FIGS. 6A-6C show X-Fuc activity staining of the mammary-gland freezesections of transgenic mice and normal mice during the lactation period.Transgenic female mice and normal female mice were sacrificed on the14^(th) day of their lactation period, and their mammary-gland tissueswere taken for sectioning. Activity staining was performed on themammary-gland sections of the transgenic mice (FIG. 6A and FIG. 6B) andnormal mice (FIG. 6C) with the X-Fuc chromogen. The results showed thatboth LPH and ecLPH of the transgenic mice were active. Also, the ecLPHappearing in the activity-stained sections of FIG. 6B proved itsextracellular property.

FIGS. 7A-7D show the results obtained by examining the lactose-degradingability of the human ecLPH enzyme, which was expressed from the mammarygland-specific expression vector of the present invention and secretedinto the milk of the transgenic animals during lactation.

FIG. 7A shows standard samples of glucose, the monosaccharide molecule,and lactose, the disaccharide molecule, established by HPLC. FIG. 7Bshows an HPLC separation curve of lactose in the milk sample from anormal ICR mouse, used as the normal control of the experiment inExample 6. FIG. 7C shows an HPLC map of the milk sample from an ecLPHtransgenic mouse, showing that 50% of the lactose was degraded toglucose. FIG. 7D shows an HPLC map of the milk sample from anon-transgenic mouse, proving that the milk contained only lactose.

FIG. 8 shows a comparison of the youngling growth curves of ecLPHtransgenic mice and normal ICR mice in the lactation period. In eachbrood of the normal ICR mice and transgenic mice, the subject numbers ofthe younglings were controlled between 8 to 10. The weights of theyounglings of the normal ICR mice and transgenic mice were measuredevery two days from the 4^(th) day after birth. Statistical data showedthat the two groups had a significant difference of p<0.001 during the4^(th) to 20^(th) days after birth.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a transgenic animal producing low-lactosemilk, wherein the transgenic animal is transformed with a novel geneencoding an extracellular lactase-hydrolyzing enzyme cloned from a humansmall intestinal cDNA library according to the present invention.According to an embodiment of the present invention, the gene encodingan extracellular human lactase-hydrolyzing enzyme of the presentinvention is preferably an extracellular human lactase-phlorizinhydrolase gene, more preferably the new extracellular humanlactase-phlorizin hydrolase gene of the present invention (ecLPH). Thenewly identified ecLPH cDNA clone containing a secreting signal peptidein the N-terminus and lacking a transmembrane domain in the C-terminuswas verified as encoding a membrane-anchorless extracellular protein inthis invention when compared to the known human lactase-phlorizinhydrolase (hLPH) gene. Since the extracellular lactase-hydrolyzingenzyme gene is constructed in a mammary gland-specific expressionvector, after being introduced into an animal, the transgene can becontinuously and stably expressed during lactation, producing largeamounts of proteins with the activity of degrading lactose. Theexpression of the heterologous gene in tissues of the mammary gland doesnot affect the normal physiological functions of the transgenic animal.The transgene specific for mammary gland expression even lacks anyadditional selection gene for antibiotic resistance. Further, mammalstransformed by the technology of the present invention breed filialgenerations carrying the heterologous transgene, with amounts ofexpression comparable to their transformed parental generation and thuscan be used to produce low-lactose milk.

The present invention also provides a method for producing a transgenicanimal that produces low-lactose milk, wherein the animal is transformedwith a gene encoding an extracellular lactase-hydrolyzing enzyme,preferably an extracellular lactase-phlorizin hydrolase gene, morepreferably the extracellular lactase-phlorizin hydrolase gene of thepresent invention (ecLPH). The newly identified ecLPH cDNA clonecontaining a secreting signal peptide in the N-terminus and lacking atransmembrane domain in the C-terminus was proposed as amembrane-anchorless extracellular protein in this invention whencompared to the known human lactase-phlorizin hydrolase (hLPH) gene. Theinventors have clearly demonstrated that the exogenic ecLPH protein canbe secreted into the mammary alveolar cavity (FIG. 6B) of lactatingtransgenic mice. In contrast, the exogenic hLPH protein can only befound in the membrane portion of the mammary gland (FIG. 6A) oflactating transgenic mice.

According to an example of the present invention, the animal wastransformed with the heterologous gene expression vector construct ofthe present invention.

Another embodiment of the present invention also provides a novelextracellular human lactase-phlorizin hydrolase (ecLPH) gene, which wascloned from a human small intestinal cDNA library and can be expressedwith a mammary gland-specific expression vector. The ecLPH cDNA of thepresent invention has a full length of 4.2 kb, and encodes anextracellular protein without a membrane-binding domain. Compared withthe gene sequence of the known mature human lactase-phlorizin hydrolase(hLPH) (FIG. 2, top panel), the ecLPH cDNA of the present invention(FIG. 2, bottom panel) lacks Exon 12 and Exon 15-17, which completelyspliced out the transmembrane domain. The coding region structure of theecLPH cDNA of the present invention is shown in FIG. 2. The newlyidentified ecLPH cDNA clone containing a secreting signal peptide in theN-terminus and lacking a transmembrane domain in the C-terminus wasverified as a membrane-anchorless extracellular protein in thisinvention.

According to an embodiment of the present invention, the extracellularhuman lactase-phlorizin hydrolase gene of the present invention has anucleotide sequence encoding the amino acid sequence as shown in SEQ IDNO: 2.

According to a preferred embodiment of the present invention, theextracellular human lactase-phlorizin hydrolase gene of the presentinvention has the nucleotide sequence as shown in SEQ ID NO: 1, or adegenerate sequence or a complementary sequence thereof.

In another embodiment, the present invention relates to a heterologousgene expression vector construct comprising the gene of the presentinvention that can be expressed with a mammary gland-specific expressionvector. More specifically, the construct comprises four parts: (1) apromoter 5′-regulatory sequence, which is specific for expression in themammary gland epithelial cells and can regulate the heterologous gene sothat the gene is continuously and stably expressed during the lactationperiod of the transgenic mammal; (2) a mammary gland-specificextracellular signal peptide sequence, which can guide the heterologousproteins expressed in the mammary gland epithelial cells of thetransgenic mammal so that the proteins are efficiently secreted into themilk; (3) a heterologous gene, which is downstream of and regulated bythe 5′-regulatory sequence and the extracellular signal peptide; and (4)a 3′-regulatory sequence, which is downstream of the coding sequence ofthe heterologous gene, comprising a polyadenylation signal sequence forthe stability and integrity of the mRNA molecule transcripted from theheterologous gene and post-transcriptional modification. According to anembodiment of the present invention, the novel extracellularlactase-phlorizin hydrolase (ecLPH) gene of the present invention isconstructed downstream of the mammary-gland specific promoter, followedby the polyadenylation signal sequence (0.5 kb) of a bovine growthfactor gene to form a mammary gland-specific mammalian vector. The mapof the transgene construct is shown in FIG. 3A.

A further embodiment of the present invention relates to a method forproducing low-lactose milk, wherein the heterologous gene expressionvector construct of the present invention is introduced into apronucleus to produce a transgenic animal that produces low-lactosemilk. According to an example of the present invention, the milk of thetransgenic animal showed that the heterologous gene was highly expressedin the mammary gland, and the transgene was continuously and stablyexpressed during lactation to produce large amounts of proteins with theactivity of degrading lactose. This proved that the transgenic animalcould indeed be used to produce low-lactose milk.

In addition, it was surprisingly found in the present invention thatwhen transgenic female mice of the present invention mated during thesame period with normal ICR female mice and fed their pups with the milksecreted after their pregnancy and delivery, the weights of pupsreceiving milk from the LPH transgenic mice increased more than those ofthe normal ICR control group, with a statistically significantdifference (p<0.001) (shown in FIG. 8). This result not only showed thatthe novel ecLPH protein had a function of degrading lactose, but alsoindicated that the transformed monosaccharide components (i.e., glucoseand galactose) could be digested more easily and might provideassistance in intestinal digestion and absorption. The result alsoindicated that the ability to lactate was raised due to a change of therole of lactose in balancing the osmotic pressure. Accordingly, thepresent invention also provides a method for increasing the growth rateand weight gain of a newborn animal, comprising transforming a maternalanimal with the extracellular lactase-phlorizin hydrolase gene of thepresent invention, and feeding the newborn animal with the milk secretedafter pregnancy and delivery of the transformed animal, preferably fromthe pregnant transgenic animal.

The aforementioned animals refer to mammals, including but not limitedto mice, cows, goats and sheep, preferably cows, goats and sheep.

The following examples are provided for further illustrating the presentinvention, but not for limiting the present invention.

EXAMPLE 1 Screening for a Novel Extracellular Lactase-PhlorizinHydrolase (ecLPH) Gene in a Human Small Intestine 5′-Stretch cDNALibrary

The screening was conducted with small fragments of specific LPHnucleotides in a human small intestine 5′-stretch cDNA library. Thesmall fragments of specific human LPH nucleotides were obtained from theGenBank of NCBI Sequence Viewer (Accession Number: X07994) for thedesignation of primer sets to generate a probe used in cDNA libraryscreening. The primers used in screening for LPH cDNA are: Primer 1:(SEQ ID NO:3) 5′-CTCTCTTGTCATCCTCTTCC-3′; and Primer 2: (SEQ ID NO:4)5′-AACAAGTCAATCAAGGCAGG-3′.

The primers used in screening for ecLPH cDNA are: Primer 3: (SEQ IDNO:5) 5′-CAGACTTTTGTTTCCAGACC-3′; Primer 4: (SEQ ID NO:6)5′-TCAACAACACGAACAGGC-3′; and Primer 5: (SEQ ID NO:7)5′-ACAGAAATGCCAAGCCACAG-3′.

In the first round, a total of 5×10⁴ pfu phages were screened. DevelopedX-ray films were properly cut and oriented with the phage culture platesto check for hybridization signals on the X-ray films. Only thoseculture plates with overlapping hybridization signals on the X-ray filmsdeveloped from two duplicate nitrocellulose (NC) films were selected. Atotal of five hybridization signal spots were screened out in the firstround, designated as 1-1, 4-1, 4-2, 4-3 and 5-1, respectively. Theconcentrations of the phages were determined by a rapid quantitativemethod using a serial dilution of phage plating on agar plates, followedby the second and third rounds of screening. The concentrations of thefive recombinant phage clones obtained after three rounds of screeningwere determined by the same rapid quantitative method. The phages wereproliferated in 150 mm culture plates to obtain a high-concentrationphage liquid. Phage DNA was extracted by phenol/chloroform purificationand ethanol precipitation, and the insert fragment was analyzed byrestriction digestion (shown in FIG. 1).

The DNA of the five recombinant phage strains was digested with EcoRI toseparate the vector from the carried hLPH cDNA fragment, and thenanalyzed by 0.8% gel electrophoresis. It was found that three of theclones carried the same hLPH cDNA fragment, which was about 6.2 kb,while the other two clones carried a cloned fragment of about 4.2 kb.The two types of clones were designated as 4-1-1-1 and 5-1-2-1,respectively, and were fully sequenced. The results of the sequencingshowed that the cloned 6.2 kb hLPH fragment (No. 4-1-1-1) encoded amembrane-anchored protein comprising a membrane-binding domain, whilethe cloned 4.2 kb ecLPH fragment (No. 5-1-2-1) encoded an extracellularprotein comprising no membrane-binding domain, which is the novel ecLPHof the present invention. The gene coding region structure of the novelecLPH cDNA is shown in FIG. 2. The various colored sections in FIG. 2 ofLPH and ecLPH (also called LPH1) mRNA structures are indicated, wherepresent, as a precursor sequence (blue box region), a maturelactase-phlorizin hydrolase domain (red box region), a transmembranedomain (green box region), and a novel 3′-UTR region (yellow boxregion). The known hLPH cDNA has a full length of 6.2 kb and encodes amembrane-anchored protein comprising a membrane-binding domain, whilethe newly cloned ecLPH (also called LPH1) cDNA has a full length of 4.2kb and lacks Exon 12 and Exon 15-17, encoding an extracellular proteincomprising no membrane-binding domain.

After sequencing, the genetic sequence was deduced for the ecLPH geneand expressed protein of the present invention. The nucleotide sequenceis SEQ ID NO: 1 and the amino acid sequence is SEQ ID NO: 2.

EXAMPLE 2 Production of Transgenic Mice and Dairy Goats Carrying theLactase-Phlorizin Hydrolase (ecLPH) Gene

According to the present invention, to breed a transgenic mammal, theextracellular human lactase-phlorizin hydrolase (ecLPH) gene wasconstructed into a mammary gland-specific alpha-lactalbumin (α-LA)promoter (2.0 kb) by DNA ligation reaction, followed by thepolyadenylation signal sequence (0.5 kb) of a bovine growth factor gene(bGH polyA) to form a mammary gland-specific mammal vector. A map of thetransgene construct is shown in FIG. 3A. The various colored sections inFIG. 3A of the transgene map indicated a mammary gland-specific α-LApromoter (dark blue box region), a secretion signal peptide (green boxregion), a mature lactase-phlorizin hydrolase domain (domain III and IVcolored by pink box region), and a novel 3′-UTR region (light blue boxregion). The vector constructed in a plasmid as stated above wasamplified by shaking the culture in 500-ml volume of bacterial hostcells and purified by plasmid DNA extraction in lysozyme and alkalireagent. A transgene construct of high purity was obtained following apreparation process comprising double digestions of HindIII and XbaIrestriction enzymes and CsCl-gradient ultracentrifugation for DNAbanding processing. The transgene was mixed at a concentration of 1-2ng/μl into a suitable microinjection buffer (10 mM Tris-HCl, 0.1 mMEDTA; pH 7.4), and injected into a male pronucleous of =mammal one-cellstage embryos by the microinjection technique. The embryos were thentransferred into the uterus of a female mammal. For the animal subjectsborn after pregnancy, rapid PCR screening and Southern blotting wereperformed on their tissue DNA to analyze the insertion pattern of theheterologous transgene in the animal genome. The results are shown inFIG. 3B and FIG. 3C. One strain of transgenic dairy goats and 7 strainsof transgenic mice were obtained by this method.

The nucleotide sequencing analysis showed that there was only one BamHIrestriction site located near the 3′-end of the ecLPH transgene (FIG.3A). Therefore, the insertion and possible concatamerizing patterns ofthe LPH transgene in the chromosomes of each mouse were determined bySouthern blot hybridization. Based on the resulting signals, it wasdetermined that the heterologous gene was inserted in the mouse genomewith various concatamerizing patterns. The concatamerizing patterns ofthe heterologous gene included head-to-head, tail-to-tail andhead-to-tail concatamerization. Moreover, the heterologous genes inparental transgenic mice No. 1 and No. 3 were passed to their offspringwithout difference in the concatamerizing pattern (left blot in FIG.3B). As for parental transgenic mouse No. 5, the heterologous geneexisted in the genome of its offspring mostly in a concatamerizingpattern wherein 5.5 kb was missing, except that No. 102 received thecomplete heterologous gene (right blot in FIG. 3B). These results showedthat the heterologous genes in all of the three strains could becompletely passed through the germ-line transmission.

EXAMPLE 3 Quantitative Analysis of Milk of the Transgenic Female Mice

Milk samples of the transgenic female mice of Example 2 were collectedfor quantitative analysis. A glass pipette sealed by mineral oil wasused to collect milk samples. Milk was drawn from anesthetizedtransgenic female mice, which were treated with oxytocin, by squeezingmassage. Milk samples were collected on the 7^(th) and 14^(th) days. Themilk of No. 1 (F₀; female) and No. 3 (F₀; female) of the parentalgeneration, and that of No. 136 and No. 137 of the filial generation(offspring of No. 5 (F₀; male)) were sampled during their lactation.Since mouse milk contains large amounts of casein and lipid, thecollected milk samples were first diluted 20-fold with deionized water,and then centrifuged at 3,000 rpm for 10 minutes at 4° C. The wheyportion was taken so that the lipid would not interfere with the gelelectrophoresis of proteins. The partially purified milk was subjectedto polyacrylamide gel electrophoresis (12% SDS-PAGE) for 1.5 hoursrunning time under 100 volts and stained with Coomassie blue. Theproteins on the electrophoresis gel were transferred to a PVDF cellulosemembrane to perform Western blotting. A rabbit anti-LPH antibody wasused to examine the amount of ecLPH proteins in the skim milk oftransgenic mice by Western blot hybridization and ELISA assays. Theresult showed that a specific signal appeared at a molecular weight of66 kD. The signal was not observed in normal ICR mice of the samestrain. Therefore, the exogenic human ecLPH protein was indeed expressedin the mammary gland and secreted in the milk of transgenic animals(FIG. 4). Using β-casein as an internal control to obtain a quantitativecalibrated value, it was determined that the amount of the exogenichuman ecLPH protein in the milk of transgenic mice at the 14^(th) day ofthe lactating period was much higher than that in the milk of the 7^(th)day. The highest amount of the exogenic human ecLPH protein detectedfrom the milk of the transgenic animals was 780 μg/ml. It was clearlydemonstrated that the newly cloned ecLPH gene encoded an extracellularsecreting ecLPH protein.

EXAMPLE 4 Immuno-Histochemistry Staining Analysis of Mammary-GlandTissue Sections of the Transgenic Female Mice

To examine the difference in the expression level of the exogenic humanecLPH protein between homozygous F₃ offspring subjects (ecLPH+/+) andheterozygous F₃ offspring subjects (ecLPH+/−), the transgenic femalemice produced according to the method of Example 2 were sacrificedduring their lactation period, and their mammary gland tissues weretaken for freeze embedding sectioning and immuno-histochemistry staining(IHC). It was clearly observed from the staining that there were redsignals resulting from AEC chromogens (peroxidase substrate) degraded byHRP (horseradish peroxidase) enzymes bound on the secondary antibodies,representing target proteins in the mammary glands of the transgenicmice, which were recognized by the specific monoclonal antibodies.Treated by the same IHC conditions, no equivalent signal was observed inthe mice of the normal ICR control group (FIG. 5). Therefore, it wasdetermined that the target protein was indeed expressed in the mammaryglands of the transgenic mice, and that homozygous transgenic femalemice had higher expression than heterozygous ones. As for the locationof expression in mammary-gland tissues, signals clearly appeared in theepithelia, alvoli cavities and lactiferous tubules. This result directlyindicated that the exogenic human ecLPH protein pertains to theextracellular form.

EXAMPLE 5 X-Fuc Activity Staining Analysis of Mammary Gland TissueSections of the ecLPH Transgenic Female Mice

To further examine the physiological enzyme activity of the ecLPHproteins in the milk of the transgenic animals, both transgenic femalemice and normal ICR female mice were sacrificed on the 14^(th) day oftheir lactation period, and their mammary-gland tissues were taken forfreeze embedding sectioning and activity staining with an LPH-specificX-Fuc chromogen substrate. The chromogen substrate is an analog of5-bromo-4-chloro-3-indolyl β-D-galactoside (X-gal), differing in thatX-Fuc is specifically degraded by the lactase-phlorizin hydrolase domainof LPH enzyme to produce a signal of a blue degraded product. Thetissues were fixed on slides with 4% paraformaldehyde in roomtemperature for 30 minutes and washed 3 times with phosphate bufferedsaline (PBS). The slides with cells attached and fixed thereon werecovered with 1 mM X-Fuc (the lactase-specific chromogen substrate)dissolved in 1 M maleate buffer (pH6.5) containing 0.5 mMp-chloromercuribenzoate, 0.05 M potassium ferricyanide and 0.05 Mpotassium ferrocyanide, and carefully put in a chamber with sufficientmoisture. The slides were left overnight at 37° C. for reaction, andexcessive fluid resulting from the reaction was removed. The slides werewashed twice with PBS, covered with 80% glycerin and sealed. The slideswere observed under a microscope for coloration, and photographed with adigital camera for recording. In FIG. 6B, blue signals resulting fromdegradation of the X-Fuc substrate clearly appeared in the alveolicavity of the mammary gland of the ecLPH transgenic mice, while no bluesignal was generated at the same location of the control group of normalICR female mice (FIG. 6C). In contrast, we also generated a controltransgenic mice harboring the prior art membrane-anchored LPH gene inthe same mammary expression vector to compare their protein localizationand enzyme activity in the mammary gland. The blue stain of degradationof X-Fuc substrate was only presented near the epithelial layer in themammary gland of the LPH transgenic mice as shown in FIG. 6A.

EXAMPLE 6 Examination of Lactose-Degrading Ability of ecLPH Enzymes inthe Milk of the Transgenic Animals

It was determined from the results obtained in the previous examplesthat ecLPH could be highly expressed in the mammary gland, and theactivity thereof has been proved with X-Fuc. However, direct evidenceproving the degradation ability of ecLPH is still required forconfirming if ecLPH can completely decompose the lactose in milk. Whenlactose is decomposed, two monosaccharide molecules, galactose andglucose, are generated. Therefore, except for directly proving thedegradation of lactose, detecting the increase in the amounts ofgalactose and glucose can also be utilized to confirm if theheterologous hLPH1 resulting from the expression of the ecLPH gene candecompose lactose, the major disaccharide molecule in milk. In thepresent invention, standard samples of glucose, the monosaccharidemolecule and lactose, the disaccharide molecule were established by HPLCanalysis for comparison with the test samples (FIG. 7A). In the HPLCseparation curve of the milk sample from a normal ICR mouse (FIG. 7B),only the peak of lactose appeared, which was used as the control ofnormal mouse milk in the present experiment. In the HPLC map of the milksample from the ecLPH transgenic mouse (FIG. 7C), the peaks of bothlactose and glucose clearly appeared. This result showed that 50% of thelactose was degraded into glucose. As for the HPLC map of the milksample from a non-transgenic mouse (FIG. 7D), only one peak appeared,proving that the milk contains only lactose, the same as that of thenormal ICR mouse.

EXAMPLE 7 Comparison of the Growth Curves of ecLPH Transgenic Mice andNormal ICR Mice

Transgenic female mice and normal ICR female mice were bred at the sametime. The 4^(th) day after the delivery was the first day of measuringthe weights of the filial mice. Afterwards, the weight of each of thefilial mice was measured every two days. The results of the weightsmeasured on the 4^(th) day and thereafter showed that the filial micereceiving milk from these LPH transgenic mice of the present invention(n=26) increased more than the weight of the normal ICR control group(n=19), with a statistically significant difference of p<0.001 (shown inFIG. 8). Such results not only showed that the novel ecLPH protein had afunction of degrading lactose, but also suggested that the transformedmonosaccharide components (i.e., glucose and galactose) could bedigested more easily and might provide assistance in intestinaldigestion and absorption. The results showed that the ecLPH-containingmilk can increase the growth rate and weight gain of newborn animals bypredigestion of lactose in their nursing milk.

The present invention successfully cloned the novel extracellularlactase-phlorizin hydrolase (ecLPH) gene, and transformed mammals likedairy goats and mice with the human ecLPH gene. The transgene can becontinuously and stably expressed during lactation, producing largeamounts of proteins having the activity of degrading lactose. Theexpression of the heterologous gene in tissues of the mammary gland doesnot affect the normal physiological functions of the transgenic animal.The transgene specific for mammary gland expression even lacks anyadditional selection gene for antibiotic resistance. Further, mammalstransformed by the technology of the present invention breed filialgenerations carrying the heterologous transgene, with amounts ofexpression comparable to their transformed parental generation and thuscan be used to produce low-lactose milk.

Without departing from the spirit and scope of the present invention, inview of the present disclosure, anyone skilled in the art may makevarious changes and modifications to introduce different heterologousgenes, which falls within the protected scope of the present invention.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. An isolated nucleic acid molecule comprising an extracellularlactase-phlorizin hydrolase gene, wherein the gene encodes a humanextracellular lactase-phlorizin hydrolase protein having nomembrane-binding domain, and the gene lacks Exon 12 and Exons 15-17compared with a known human lactase-phlorizin hydrolase (hLPH) gene. 2.The isolated nucleic acid molecule of claim 1 comprising a nucleotidesequence encoding an amino acid sequence of SEQ ID NO:
 2. 3. Theisolated nucleic acid molecule of claim 1 comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence of SEQ ID NO: 1; (b) a degenerate sequence of SEQ ID NO:1; and(c) a complementary sequence of (a) and (b).
 4. An isolated humanextracellular lactase-phlorizin hydrolase protein encoded by the nucleicacid molecule of claim
 1. 5. The isolated human extracellularlactase-phlorizin hydrolase protein of claim 4 comprising an amino acidsequence of SEQ ID NO:2.
 6. The isolated human extracellularlactase-phlorizin hydrolase protein of claim 4 being a mature proteinhaving no signal peptide sequence.
 7. A mammary gland-specificexpression vector comprising a nucleic acid molecule of claim 1, whereinthe mammary gland-specific expression vector expresses a humanextracellular lactase-phlorizin hydrolase protein in a mammary gland ofa transgenic mammal transformed with the mammary gland-specificexpression vector.
 8. The mammary gland-specific expression vector ofclaim 7, comprising (a) a first nucleotide sequence encoding a mammarygland-specific extracellular signal peptide sequence; (b) a secondnucleotide sequence encoding a mature human extracellularlactase-phlorizin hydrolase protein having no membrane-binding domain,linked to the 3′-end of the first nucleotide sequence, wherein thesignal peptide sequence guides the secretion of the mature humanextracellular lactase-phlorizin hydrolase protein into milk produced bythe transgenic animal; (c) a third nucleotide sequence encoding a3′-untranslated region (3′-UTR), linked to the 3′-end of the secondnucleotide sequence, wherein the 3′-UTR comprises a polyadenylationsignal; and (d) a promoter sequence linked to the 5′-end of the firstnucleotide sequence, wherein the promoter sequence regulates thetranscription of a gene comprising the first nucleotide sequence, thesecond nucleotide sequence and the third nucleotide sequence, in mammarygland epithelial cells of the transgenic mammal during a lactationperiod of the transgenic mammal.
 9. The mammary gland-specificexpression vector of claim 8, wherein the promoter sequence comprisesalpha-lactalbumin (α-LA) promoter.
 10. The mammary gland-specificexpression vector of claim 8, wherein the second nucleotide encodes amature human extracellular lactase-phlorizin hydrolase proteinconsisting of domain III and IV of the human extracellularlactase-phlorizin hydrolase protein.